Insulin binds to its receptor on the surface of the target cell, thereby activating the insulin receptor tyrosine kinase. The activated receptor phosphorylates tyrosine residues in multiple intracellular proteins. We have investigated the downstream pathway that eventuates in translocation of GLUT4 glucose transporters to the plasma membrane in isolated rat adipocytes, a physiologically important target cell for insulin action. Using an technique involving transient expression in rat adipocytes, we have previously demonstrated that IRS-1 is one of the substrates which participates in mediating the effect of insulin to translocate GLUT4, and that activation of phosphatidyl inositol 3-kinase (PI 3-kinase) activity also plays a necessary role in mediating this action of insulin. Recently, we have extended these studies in several ways: (1) characterizing interactions of IRS-3 with receptor tyrosine kinases and also with downstream signaling molecules; (2) cloning and characterization of a novel insulin receptor substrate protein in Drosophila. In addition, we have initiated studies to define the subcellular localization of IRS proteins, and also to define the sequences that are responsible for targeting of IRS to its normal location within the cell.